Machado-Joseph Disease (MJD) is a dominantly inherited neurodegenerative disease characterized by a loss of muscle coordination, for which currently only symptomatic treatments exist. MJD is a CAG/polyglutamine disorder, in which patients possess expanded CAG nucleotide repeats in the ATXN3 gene. This results in an abnormally long polyglutamine tract, as well as misfolding and aggregation of the ataxin-3 protein. Since reducing levels of disease proteins improves pathology in animal models of polyglutamine diseases, reducing levels of pathogenic ataxin-3 could be a viable therapy for MJD. We recently demonstrated that ataxin-3 does not require ubiquitination to be degraded. Rather, its turnover is regulated by ubiquitin-binding site 2 (UbS2) on its N terminus. Ataxin-3 is stabilized by its interaction with the proteasome-associated proteins Rad23A/B through UbS2. Mutating UbS2 to disrupt the interaction between ataxin-3 and Rad23A/B decreases ataxin-3 protein levels in cultured mammalian cells. These findings led us to conclude that UbS2 could be a potential target through which to enhance ataxin-3 degradation for MJD therapy. To test this hypothesis, first, we propose to determine the molecular mechanism by which ataxin-3 is degraded in vitro. To accomplish this, we will carry out a panel of in vitro reactions with differet combinations of wild type or mutated ataxin-3, proteasomes, and Rad23A/B. We will confirm our findings from in vitro recombinant assays by examining molecular complexes in mammalian cells through co-immunoprecipitations and size-exclusion chromatography. Secondly, we will test our hypothesis for ataxin-3 degradation in vivo by generating transgenic Drosophila lines that express either ataxin-3 with all of its domains intact, or ataxin-3 with UbS2 mutated. We will determine the role of Rad23 and UbS2 in ataxin-3-dependent toxicity through tissue-specific expression of our constructs. We will examine lethality, longevity, morphology, histology, and motility in these flies. Finally, we will conduct a discovery-based screen for novel suppressors of ataxin-3 dependent degeneration in vivo, utilizing a method that we developed in our laboratory, which reports retinal integrity in intact flies by using membrane-bound GFP. The goals of this proposed research coincide with the mission of the National Institute of Neurological Disorders and Stroke, as the results from this research can be translated into efficacious therapies for patients suffering from MJD.